【Title】

“Culture and identification of endothelial progenitor cells from human umbilical cord blood in vitro”living pictures

【Source】

Journal of Clinical Rehabilitative Tissue Engineering Research  November 5, 2010  Vol.14 No. 45，8450-8454

【Author】

Fang Li-jian¹, Song E², Luan Ying², Lu Cheng-wei², Yang Wei², Shi Hui², Bi Ming-chao²

【Setting】

¹Department of Ophthalmology, Fangshan District Liangxiang Hospital, Beijing  102401, China; ²Department of Ophthalmology, First Clinical Hospital, Jilin University, Changchun  130021, Jilin Province, China

【Found】

Supported by: the Doctor Center Foundation of Jilin Province, No. 2007018311*; the International Cooperation Program of Committee of Science of Jilin Province, No. 20050705-3*

【Abstract】

BACKGROUND: The method of traditional immunomagnetic beads sorting endothelial progenitor cells (EPCs) shows complicated operation, with high cost and low cell obtaining rate. Moreover, the proliferation and differentiation of EPCs are limited.

OBJECTIVE: To elucidate a method for in vitro induction and identification of EPC from human cord blood monocytes (CBMC), to realize EPC transplantation, and to provide enough cell source for improving blood vessel function.

METHODS: CBMCs were isolated by Percoll density gradient centrifugation from cord blood, in vitro induced to differentiate and amplified, and then identified by immunohistochemical and immunofluorescent staining and flow cytometry. 

RESULTS AND CONCLUSION: During culture, the cells became spindle-shaped and displayed cobble-stone morphology with outgrowth. On day 7, immunostaining of adherent cells was positive for the cell markers CD31 and vWF. (83.0±4.3)% of attached cells were positive for the double marker DiI-acLDL/FITC-UEA-Ⅰ. The red fluorescence of DiI-acLDL-labeled EPCs lasted for over 6 weeks in vitro. On day 7, flow cytometric analysis showed positive staining of attached cell for CD34, CD133 and KDR (17.8±3.7)%, (22.1±4.4)% and (81.5±5.0)%, respectively. Results indicate that the mononuclear cells from CBMC differentiate into EPCs. It can differentiate into endothelial cells by in vitro amplification.

【JPG pictures】

4 figures click

Figure 1  Observation of cultured cells under an inverted microscope

Figure 2  Immunohistochemical method for 3,3'-diaminobenzidine on d 7 following cell culture, adherent cells show brow color (×200)

Figure 3  On d 7 following cell culture under a laser scanning confocal microscope (×200)

Figure 4  At d 7, expression rate of CD133, CD34 and KDR using flow cytometry

【Full text】